TWI441648B - Foxp3 peptide vaccine - Google Patents

Description

FOXP3 peptide vaccine [related application]

This application is based on US Provisional Application No. 60/878,615, filed on Jan. 3, 2007, and U.S. Provisional Application No. 60/896,472, filed on March 22, 2007. The complete teachings of these applications are incorporated herein by reference.

The present invention relates to the field of biological sciences, and in particular to the field of cancer treatment. In particular, the present invention relates to a Foxp3 peptide which is extremely useful as a cancer vaccine and relates to a medicament for treating and preventing a tumor.

It has been demonstrated that CD8 ⁺ cytotoxic T lymphocytes (CTLs) recognize epitope-derived peptides derived from tumor-associated antigens (TAAs) present on MHC class I molecules, and then kill the tumor cells. Since the discovery of the MAGE family as the first TAA, many other TAAs have also been discovered using immunological methods (Boon T, Int J Cancer 54: 177-80, 1993; Boon T et al., J Exp Med 183: 725- 9, 1996; van der Bruggen P et al., Science 254: 1643-7, 1991; Brichard V et al., J Exp Med 178: 489-95, 1993; Kawakami Y et al., J Exp Med 180: 347 -52, 1994), some of which have entered the clinical development phase as targets for immunotherapy.

Identifying new TAAs that induce potency and specific anti-tumor immune responses is the basis for further development of clinical peptide vaccination strategies for various types of cancer (Harris CC, J Natl Cancer Inst 88: 1442-5, 1996; Butterfield LH et al., Cancer Res 59: 3134-42, 1999; Vissers JLM et al., Cancer Res 59: 5554-9, 1999; Van der Burg SH et al., J Immunol 156: 3308-14, 1996; Tanaka F et al., Cancer Res 57: 4465-8, 1997; Fujie T et al., Int J Cancer 80: 169-72, 1999; Kikuchi M et al., Int J Cancer 81: 459-66, 1999; Oiso M et al., Int J Cancer 81: 387-94, 1999).

A number of antigen-specific immunotherapies have been implemented, however, in terms of significant tumor regression, only low clinical efficacy has been obtained to date (Rosenberg SA et al., Nat Med 10:909-15, 2004). One of the important reasons is that the immune response to tumor-through lymphocytes (TIL) and peripheral blood lymphocytes (PBL) is poor for patients with cancer at the advanced stage (Miescher S et al., J Immunol 136: 1899- 907, 1986). This immunosuppression induced by tumors is poor response to tumor antigens (Young RC et al., Am J Med 52: 63-8, 1972), poor T cell proliferation (Alexander JP et al., Cancer Res 53: 1380) -7, 1993), loss of cytokine production (Horiguchi S et al., Cancer Res 59: 2950-6, 1999), and defective signaling by T cells and natural killer cells (Kono K et al., Clin Cancer Res 11: 1825-8, 1996, Kiessling R et al., Cancer Immunol Immunother 48: 353-62, 1999).

In order to improve the clinical efficacy of immunotherapy, it is important to overcome the effects of tumor-induced immunosuppressive factors. Immune tolerance and protection from autoimmunity, from central and peripheral mechanisms These mechanisms include the deletion of clonal reproductive lines of autoreactive T cells in the thymus and the induction of sensitization in the presence of autoantigens in the periphery. Recently, regulatory T cells (T-reg) characterized by co-expression of CD4 and CD25 have been devised, and are functionally unique groups of T cells whose function is to maintain constant immunity (Sakaguchi S et al., J Immunol. 155 : 1151-64, 1995, Dieckmann D et al., J Exp Med 193: 1303-10, 2001). T-reg cells are one of the major inhibitors of various types of immune responses (Miescher S et al., J Immunol 136: 1899-907, 1986; Young RC et al., Am J Med 52: 63-72, 1972; Alexander JP et al., Cancer Res 53: 1380-7, 1997; Horiguchi S et al., Cancer Res 59: 2950-6, 1999; Kono K et al., Clin Cancer Res 11: 1825-8, 1996; Kiessling R et al., Cancer Immunol Immunother 48: 353-62, 1999).

Although the key inter-molecular interactions and message transmission pathways for the production of T-reg and its functions are not fully understood, T-reg requires a forkhead encoded by the Foxp3 gene (GenBank Accession No. NM_014009; SEQ ID NO 1). The transcription factor scurfin (Foxp3; SEQ ID NO 2), whose transcription factor scurfin controls the developmental and regulatory properties of this (Fontenot JD et al., Nat Immunol 4: 330-6, 2003, Hori S et al., Science 299 : 1057-61, 2003, Khattri R et al., Nat Immunol 4: 304-6, 2003). Furthermore, mice were vaccinated with dendritic cells transfected with Foxp3 mRNA to elicit a Foxp3-specific CTL response (Smita N et al., Cancer Res. Jan 1;67(1):371-80, 2007).

Therefore, Foxp3 is the target of cancer immunotherapy, and further, a part of the peptide encoded by Foxp3 can be used as an antigen recognized by CTL.

In order to improve the clinical efficacy of immunotherapy, it is important to overcome the immunosuppressive factors induced by tumors. T-reg has been found to be one of the important roles in suppressing multiple types of immune responses. Therefore, it is important to develop a vaccine targeting T-reg expressing Foxp3 to overcome T-reg-induced immunosuppression.

The present invention is based on the identification of an epitope peptide derived from the gene product of Foxp3, which elicits a cytotoxic T lymphocyte (CTL) specific to the Foxp3 peptide or epitope. Peripheral blood mononuclear cells (PBMC) from healthy providers were stimulated with HLA-A*24 and HLA-A*02 binding candidate peptides from Foxp3. These peptides have been shown to be HLA-A24 or HLA-A02-restricted epitope peptides that are potent against T-reg-expressing potent and specific immune responses that express Foxp3.

In summary, the present invention provides a method for modulating immunosuppression comprising the step of administering a Foxp3 multipeptide of the present invention. For example, anti-immunosuppression (i.e., reversal or offsetting immunosuppression) of cytotoxic T lymphocytes is induced by administration of the Foxp3 polypeptide. Accordingly, the present invention provides a method for inducing anti-immunosuppression comprising the steps of administering the Foxp3 multi-peptide, and providing a pharmaceutical preparation for modulating immunosuppression, the pharmaceutical preparation comprising the Foxp3 multi-peptide.

In one aspect of the invention, the invention provides a peptide comprising or consisting of the amino acid sequences selected from the group consisting of SEQ ID NOs: 3-5, 7-9, 12, 15- 19, 22, 24, 27-30, 37, 67 or 74.

In another aspect of the invention, the invention provides a peptide having cytotoxic T cell inducing ability, wherein the peptide comprises or consists of an amino acid sequence selected from the group consisting of: (a) SEQ ID NO : 3-5, 7-9, 12, 17, 67 or 74; and (b) SEQ ID NO: 3-5, 7-9, 12, 17, 67 or 74, wherein one, two or several amine groups The acid is substituted or added.

In another aspect, the invention provides a peptide having cytotoxic T cell inducing ability, wherein the peptide comprises an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 15-19, 22, 24, 27-30 or 37, and (b) SEQ ID NO: 15-19, 22, 24, 27-30 or 37, wherein 1, 2 or several amino acids are substituted or added.

With respect to these embodiments, in some embodiments, the second amino acid is phenylalanine, tyrosine, methionine or tryptophan from the N-terminus. In some embodiments, the C-terminal amino acid is phenylalanine, leucine, isoleucine, tryptophan or methionine. In some embodiments, the second amino acid from the N-terminus is leucine or methionine. In some embodiments, the C-terminal amino acid is valine or leucine. For example, the substituted peptide comprises the amino acid sequence of SEQ ID NO: 95, 97 or 98.

The present invention further provides a composition comprising the Foxp3 peptide of the present invention, or a polynucleotide encoding the Foxp3 peptide of the present invention, and a pharmaceutically acceptable Accepted by the carrier or excipient. In some embodiments, the compositions are formulated to be administered as a vaccine.

The compositions may comprise one or a plurality of different peptides of the Foxp3 peptide of the invention. These compositions are useful for inhibiting T-reg cells, such as inhibiting proliferation or suppressing the function of T-reg cells.

In some embodiments, the compositions comprise more than one Foxp3 peptide, the one or more Foxp3 peptides elicit an immune response, and the T-reg cells in an individual that inhibits the HLA antigen to be HLA-A24. In some embodiments, the compositions comprise more than one Foxp3 peptide, the one or more Foxp3 peptides elicit an immune response, and the T-reg cells in an individual that inhibits the HLA antigen to be HLA-A02.

In another aspect, the invention provides a composition comprising a polynucleotide encoding a Foxp3 peptide of the invention. In some embodiments, the compositions comprise a majority (i.e., 2 or more) polynucleotides encoding a majority of the Foxp3 peptides of the invention. In some embodiments, the compositions comprise a polynucleotide encoding a majority of a Foxp3 peptide of the invention.

In some embodiments, the compositions comprise an additional peptide having the ability to induce cytotoxic T cells against cancer cells, or other polynucleotides encoded as other peptides.

In another aspect, the invention provides an exosome having a complex on its surface, the complex comprising an HLA antigen and a Foxp3 peptide of the invention. In some embodiments, the HLA antigen is selected from the group consisting of HLA-A24, HLA-A2402, HLA-A02, and HLA-A0201.

In a related aspect, the present invention provides a method for treating cancer (for example, reducing tumor cell growth and promoting tumor cell death) by administering a Foxp3 peptide or a multinuclear encoding a Foxp3 peptide to one body. Glycosylate.

In another aspect, the present invention provides a method of inducing an antigen-presenting cell which exhibits a high cytotoxic T cell inducing ability by administering a Foxp3 peptide of the present invention or encoding the Foxp3 peptide. Polynucleotide.

In another aspect, the invention provides a method of inducing cytotoxic T cells by administering a Foxp3 peptide of the invention or a polynucleotide encoding the Foxp3 peptide.

In a related aspect, the present invention provides an isolated cytotoxic T cell which is induced by the Foxp3 peptide of the present invention.

In another aspect, the invention provides an antigen presenting cell comprising a complex formed between an HLA antigen and a Foxp3 peptide of the invention. In some embodiments, the antigen exhibits cell isolation.

In another aspect, the invention provides a method of modulating T-reg cells in a body comprising administering to the individual a vaccine comprising a Foxp3 peptide of the invention or an immunologically effective peptide A fragment, or a polynucleotide encoded as one of the peptides.

In practicing the methods of treatment of the invention, the individual or patient can be a human.

The above summary of the invention and the exemplary embodiments set forth below are not intended to limit the invention.

I. Definition

The use of "a" or "an", unless otherwise specified, means "at least one".

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of an amino acid residue. The term applies to amino acid polymers in which one or more amino acid residues are modified residues or non-naturally occurring residues, such as artificial chemical analogs corresponding to naturally occurring amino acids, and naturally occurring Amino acid polymer.

The term "amino acid" as used herein, refers to naturally occurring and synthetic amino acids, and amino acid analogs and amino acid mimetics have similar functions to naturally occurring amino acids. The naturally occurring amino acid is encoded by the genetic code and is modified in the cell after translation (eg, hydroxyproline, gamma-carboxy glutamic acid, and O-phosphoric acid). The phrase "amino acid analog" refers to a compound having the same basic chemical structure as the naturally occurring amino acid (an alpha carbon bonded to a hydrogen, a carboxyl group, an amine group, and an R group), but once Modified R group or modified backbone (eg, homocysteine, n-aminoglycolic acid, amidium thiomethionate, methyl methionine methyl salt). The phrase "amino acid mimetic" refers to a chemical compound having a different structure but a similar function to a general amino acid.

The amino acid is here indicated by its universal three letter symbol or by the letter symbol of one of the IUPAC-IUB Biochemical Nomenclature Commission recommendations.

The terms "gene", "polynucleotide", "nucleotide" and "nucleic acid" are used interchangeably herein unless otherwise specified, and are similar to amino acids, which are usually Accepted single letter code representation.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning meaning meaning In the event of inconsistency, the present specification, including definitions, will control.

II. peptide

In order to demonstrate that the peptide derived from Foxp3 acts as one of the antigens recognized by cytotoxic T cells (CTLs), in the present invention, the peptide of the subsequence of the peptide Foxp3 is analyzed to know whether it is HLA-A24 or HLA. -A02 The epitope of the antigen restricted by H02-A24 or HLA-A02 is a common HLA dual gene in the world (Date Y et al., Tissue antigen 47: 93-101, 1996; Kondo A et al. J Immuol 155: 4307-12, 1995; Kubo RT et al., J Immuol 152: 3913-24, 1994). The HLA-A24 and HLA-A02 binding peptide candidates for the Foxp3 subsequence were identified using their binding affinity information for HLA-A24 and HLA-A02. After in vitro stimulation of T cells using dendritic cells (DC) carrying these peptides, CTLs were successfully established using the following sequences: Foxp3-A24-9-363 (SEQ ID NO 3), Foxp3-A24-9-366 ( SEQ ID NO 7), Foxp3-A24-9-190 (SEQ ID NO 9), Foxp3-A24-9-207 (SEQ ID NO 4), Foxp3-A24-9-332 (SEQ ID NO 5), Foxp3- A24-9-337 (SEQ ID NO 8), Foxp3-A24-10-114 (SEQ ID NO 12), Foxp3-A2-9-390 (SEQ ID NO 15), Foxp3-A2-9-69 (SEQ ID NO 16), Foxp3-A2-9-252 (SEQ ID NO 17), Foxp3-A2-10-359 ( SEQ ID NO 22), Foxp3-A2-10-263 (SEQ ID NO 24), Foxp3-A2-10-94 (SEQ ID NO 27), Foxp3-A2-10-233 (SEQ ID NO 28), Foxp3- A2-10-152 (SEQ ID NO 29), Foxp3-A2-10-77 (SEQ ID NO 30), Foxp3-A2-10-246 (SEQ ID NO 37), Foxp3-A2-9-68 (SEQ ID NO 18), Foxp3-A2-9-304 (SEQ ID NO 19), Foxp3-A24-10-87 (SEQ ID NO 67), and Foxp3-A24-10-60 (SEQ ID NO 74).

These established CTLs show specific CTL activity against the target cells pulsed by the peptide. These results are consistent with the conclusion that Foxp3 is an antigen recognized by CTL, and Foxp3-A24-9-363 (SEQ ID NO 3), Foxp3-A24-9-366 (SEQ ID NO 7), Foxp3-A24-9 -190 (SEQ ID NO 9), Foxp3-A24-9-207 (SEQ ID NO 4), Foxp3-A24-9-332 (SEQ ID NO 5), Foxp3-A24-9-337 (SEQ ID NO 8) , Foxp3-A24-10-114 (SEQ ID NO 12), Foxp3-A2-9-390 (SEQ ID NO 15), Foxp3-A2-9-69 (SEQ ID NO 16), Foxp3-A2-9-252 (SEQ ID NO 17), Foxp3-A2-10-359 ( SEQ ID NO 22), Foxp3-A2-10-263 (SEQ ID NO 24), Foxp3-A2-10-94 (SEQ ID NO 27), Foxp3-A2-10-233 (SEQ ID NO 28), Foxp3- A2-10-152 (SEQ ID NO 29), Foxp3-A2-10-77 (SEQ ID NO 30), Foxp3-A2-10-246 (SEQ ID NO 37), Foxp3-A2-9-68 (SEQ ID NO 18), Foxp3-A2-9-304 (SEQ ID NO 19), Foxp3-A24-10-87 (SEQ ID NO 67), and Foxp3-A24-10-60 (SEQ ID NO 74) are HLA-A24 And the epitope-restricted peptide restricted by HLA-A2. Since Foxp3 is expressed in most cancer patients and is associated with immunosuppression induced by tumor-induced immunosuppressive factors, Foxp3 is a good target for immunotherapy to promote antigen-specific immunotherapy against cancer. Clinical efficacy.

Accordingly, the present invention provides a nine-peptide (a peptide consisting of 9 amino acid residues) and a ten-peptide (a peptide consisting of 10 amino acid residues). The Foxp3 peptide of the present invention binds to an HLA molecule and induces cytotoxic activity of cytotoxic T lymphocytes (CTL). More specifically, the present invention provides a peptide consisting of the following amino acid sequence populations: SEQ ID NOs: 3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 or 74.

In general, software programs are available on the Internet, as described in Parker KC. et al, J Immunol. 1994 Jan 1; 152(1): 163-75, which can be used in computer calculations (in silico). The binding affinity of various peptides and HLA antigens. The binding affinity to the HLA antigen can be measured in vitro as previously described, for example, in Parker KC. et al, J Immunol. 1994 Jan 1; 152(1): 163-75.; Nukaya I. et al, Int J Cancer 1999 Jan 5;80(1):92-7.; Kuzushima K, et al. ((2001) Blood.;98(6):1872-81.; Journal of Immunological Methods, 1995, 185: 181-190 .; Protein Science, 2000, 9: 1838-1846).

Further, the Foxp3 peptide of the present invention may be adjacent to an additional amino acid residue as long as the Foxp3 peptide retains its CTL inducing ability. Such a peptide having CTL inducing ability may be less than about 40 amino acids, such as less than about 20 amino acids, such as less than about 15 amino acids. The peptide consisting of the following amino acid sequence population: SEQ ID NO: 3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 or 74, the amine group The acid sequence is not limited and may be composed of any kind of amino acid as long as it does not inhibit the CTL inducing ability of the peptide. Accordingly, the present invention also provides a peptide having CTL inducibility, comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 3-5, 7-9, 12, 15-19, 22, 24, 27 -30, 37, 67 and 74.

In general, it is known that the modification of one or more amino acids in a protein sometimes does not affect the function of the protein and even enhances the desired function of the original protein. In fact, the modified peptide (ie, substituted, added 1, 2 or a plurality of amino acid residues to the amino acid sequence modified by the original reference sequence to form a peptide) known to retain the biological activity of the original peptide (Mark et al., Proc Natl Acad Sci USA 81 : 5662-6, 1984; Zoller and Smith, Nucleic acid Res 10: 6487-500, 1982; Dalbadie-McFarland et al., Proc Natl Acad Sci USA 79: 6409-13, 1982. Thus, in accordance with one embodiment of the present invention Morphology, a peptide having CTL inducing ability of the present invention, may be an amino acid comprising SEQ ID NO: 3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 or 74 A sequence consisting of one or more amino acids added and/or substituted.

Those skilled in the art will appreciate that changing a single amino acid or a small proportion of an amino acid, individual addition or substitution of an amino acid sequence, may conserve the nature of the original amino acid side chain; It is a "conservative substitution" or a "conservative modification" in which a protein change results in a protein having a similar function. Providing a conservative substitution table that is functionally similar to an amino acid is well known in the art. Examples of the side chain properties of amino acids are: hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E) , Q, G, H, K, S, T), and side chains with the following functional groups or common characteristics: an aliphatic side chain (G, A, V, L, I, P); hydroxyl-containing side chain (S, T, Y); side chain containing sulfur atom (C, M); side chain containing carboxylic acid and guanamine (D, N, E, Q); side chain containing alkali (R, K, H ); and aromatic side chains (H, F, Y, W). In addition, the following 8 groups contain amino acids that are mutually conservatively substituted: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E) ; 3) Aspartic acid (N), glutamic acid (Q); 4) arginine (R), lysine (K); 5) Isoleucine (I), leucine (L) ), methionine (M), proline (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), sulphamine Acid (T), and 8) cysteine (C), methionine (M) (see, for example, Creighton, Proteins (1984)).

Such a conservatively modified peptide can be considered as a peptide of the present invention. However, the peptide of the present invention is not limited thereto, and may include a non-conservative modification as long as the peptide retains the CTL inducing ability. Furthermore, the modified peptide does not exclude a peptide, an interspecies homolog, and a Foxp3 dual gene that induce a polymorphic variant of CTL.

A small number (eg, 1, 2, or several) or a small proportion of amino acid residues can be modified (addition or substitution) while still maintaining the desired CTL inducibility (ie, CTL activation). Here, the term "several" means 5 or less or, for example, 3 or less. The modified amino residue may be less than 20% of the complete amino acid sequence SEQ ID NO: 3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 and 74 For example, 15% or less, such as 1 to 5%. Foxp3 peptides having at least 95%, 96%, 97%, 98%, 99% amino acid sequence identity to the identified entire sequence are contemplated by the present invention. Sequence identity can be measured using algorithms known in the art, such as BLAST, available from the National Center for Biotechnolcgy Information (website) Ncbi.nlm.nih.gov/blast/Blast.cgi).

For the homology (ie, sequence identity) analysis of the current peptide, Foxp3-A24-9-363 (SEQ ID NO 3), Foxp3-A24-9-366 (SEQ ID NO 7), Foxp3-A24-9- 190 (SEQ ID NO 9), Foxp3-A24-9-207 (SEQ ID NO 4), Foxp3-A24-9-332 (SEQ ID NO 5), Foxp3-A24-9-337 (SEQ ID NO 8), Foxp3-A24-10-114 (SEQ ID NO 12), Foxp3-A2-9-390 (SEQ ID NO 15), Foxp3-A2-9-69 (SEQ ID NO 16), Foxp3-A2-9-252 ( SEQ ID NO 17), Foxp3-A2-10-359 (SEQ ID NO 22), Foxp3-A2-10-263 (SEQ ID NO 24), Foxp3-A2-10-94 (SEQ ID NO 27), Foxp3- A2-10-233 (SEQ ID NO 28), Foxp3-A2-10-152 (SEQ ID NO 29), Foxp3-A2-10-77 (SEQ ID NO 30), Foxp3-A2-10-246 (SEQ ID NO 37), Foxp3-A2-9-68 (SEQ ID NO 18), Foxp3-A2-9-304 (SEQ ID NO 19), Foxp3-A24-10-87 (SEQ ID NO 67), and Foxp3-A24 -10-60 (SEQ ID NO 74), showing that these do not have significant homology to the peptide derived from any other known human gene product. this The situation reduces the likelihood of an unknown or unwanted immune response when used in immunotherapy.

When used in immunotherapy, the peptide of the present invention will be presented in the form of a complex with an HLA antigen on the surface of a cell or exosome. Therefore, in addition to the CTL inducing ability, the peptide is selected with a high binding affinity for the HLA antigen. Furthermore, the peptide can be modified with amino acid residues such as substitutions, additions, etc. to achieve higher binding affinity. In addition to the naturally displayed peptide, the regularity (i.e., identity) of the peptide sequence bound to the HLA antigen is known (J Immunol 152: 3913, 1994; Immunogenetics 41: 178, 1995; J Immunol 155: 4307, 1994). Therefore, the modification of the immunogenic peptide of the present invention can be carried out in accordance with such regularity. For example, a peptide exhibiting high HLA-A24 binding affinity, the N-terminal is substituted with a second amino acid to be phenylalanine, tyrosine, methionine or tryptophan, and the C-terminal amine of the peptide is obtained. Substituting a base acid for amphetamine, leucine, isoleucine, tryptophan or methionine is also useful. On the other hand, the N-terminal is substituted with a second amino acid to be leucine or methionine, and wherein the C-terminal amino acid is substituted with a peptide of lysine or leucine, and can also be used as a peptide. High HLA-A02 binds to the peptide of affinity. The substitution is not only carried out at the terminal amino acid, but also the potential TCR recognition position in the peptide. Zaremba et al. demonstrated that the amino acid substitution in the CAP1 peptide is equivalent or better than the original (Cancer Res. 57, 4570-4577, 1997). For example, the substituted peptide comprises the amino acid sequence SEQ ID NO: 95, 97 or 98. Further, one or two amino acids may be added to the N and/or C-terminus of the peptide. Such modified peptides having high HLA antigen binding affinity may also be included in the present invention.

However, when the peptide sequence is identical to a part of the amino acid sequence of an endogenous or exogenous protein with different functions, it may induce side effects against specific substances, such as autoimmune diseases or allergy symptoms. Thus, a library of available data can be used for homology studies to avoid or minimize or minimize the occurrence of amino acid sequences in which the peptide sequence is complexed with other proteins. When homologous studies have revealed that there are no other peptides that differ from the target peptide with a mono or diamino acid, the target peptide can be modified to increase binding affinity to the HLA antigen, and/or increase CTL induces without the risk of side effects.

The peptides having high binding affinity for HLA antigens described above are very effective. According to the candidate peptide selected by using the high binding affinity as an index, it is possible to actually test whether or not the CTL inducing ability exists. Here, the phrase "CTL inducing ability" represents the ability of the peptide to induce CTL when presented on an antigen presenting cell. Further, "CTL inducibility" includes the ability of the peptide to induce CTL activation, CTL proliferation, and increase IFN-γ production.

Confirmation of CTL inducing ability can be carried out by inducing antigen-presenting cells (such as B-lymphocytes, macrophages, and dendritic cells) carrying human MHC antigens, or more specifically, from human peripheral blood samples. The dendritic cells of the nucleated white blood cells are mixed with CD8-positive cells after being stimulated with the peptide, and then the IFN-γ produced and released by the CTL against the target cells is measured. As for the reaction system, a gene-transformed animal that has been produced to express a human HLA antigen can be used (for example, described in Ben Mohamed L, Krishnan R, Longmate J, Auge C, Low L, Primus J, Diamond DJ, Hum Immunol 61 (8). : 764-79, 2000 Aug, Related Articles, Books, Linkout Induction of CTL reactoin by a minimal epitope vaccine in HLA A*0201/DR1 transgenic mice: dependence on HLA class II restricted T(H) reaction). For example, the target cells can be radiolabeled with ⁵¹ Cr or the like, and the cytotoxic activity can be calculated from the radioactivity released from the target cells. Alternatively, IFN-γ produced and released by CTL can be measured in the presence of an antigen-presenting cell with an immobilized peptide, and an inhibitory zone on the medium can be visualized using an anti-IFN-γ monoclonal antibody to examine.

As a result of the test of the CTL inducing ability of the peptide as described above, it is not necessarily highly inducible for a high binding affinity of an HLA antigen. Further, selected from the peptides comprising the amino acid sequence SEQ ID NO: 3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 or 74 Tetrapeptide, showing high CTL inducibility and high binding affinity for HLA antigen.

In addition to the above-described modification of the peptide of the present invention, the peptide of the present invention can be further linked to other substances as long as the CTL inducing ability is retained. Substances that can be used include: peptides, lipids, sugars and sugar chains, ethyl sulfonyl groups, natural synthetic polymers, and the like. The peptide may comprise modifications such as glycosylation, side chain oxidation or phosphorylation; as long as the modifications do not destroy the biological activity of the peptide described herein. Such modifications can be implemented to provide additional functionality (eg, targeting function, and delivery function) or to stabilize the multi-peptide.

For example, in order to increase the in vivo stability of a multi-peptide, it is known in the art to introduce various D-amino acids, amino acid mimetics or unnatural amino acids which are particularly useful; this concept can also be applied to The multi-peptide of the invention. Polypeptide The stability can be analyzed in several ways. For example, peptidase and various biological media, such as human plasma and serum, have been used to test for stability (see, for example, Verhoef et al., Eur J Drug Metab Pharmacokin 11: 291-302, 1986).

III. Preparation of the peptide

The peptide of the present invention can be prepared using conventional techniques. For example, the peptide can be synthesized by recombinant DNA technology or chemical synthesis. The peptide of the present invention may also be synthesized individually or as a longer multi-peptide comprising two or more peptides (for example, two or more Foxp3 peptides, or one Foxp3 peptide and one non-Foxp3 peptide). The peptide can be isolated, i.e., purified to be substantially free of other naturally occurring host cell proteins and fragments thereof, for example, purified by at least about 70%, 80%, or 90%.

The peptide of the present invention can be obtained by chemical synthesis depending on the selected amino acid sequence. For example, synthetic peptide synthesis methods which can be synthesized include: (i) Peptide Synthesis, Interscience, New York, 1966; (ii) The Proteins, Vol. 2, Academic Press, New York, 1976; (iii) Peptide Synthesis ( In Japanese), Maruzen Co., 1975; (iv) Basics and Experiment of Peptide Synthesis (in Japanese), Maruzen Co., 1985; (v) Development of Pharmaceuticals (second volume) (in Japanese), Vol. 14 (peptide Synthesis), Hirokawa, 1991; (vi) WO99/67288; and (vii) Barany G. & Merrifield R. B., Peptide Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.

Alternatively, the peptide of the present invention can be obtained by any known genetic engineering method for producing a peptide (for example, Morrison J. (1977) J. Bacteriology 132: 349-51; Clark-Curtiss & Curtiss (1983) Methods in Enzymology (eds. Wu et al.) 101: 347-62). For example, first, a suitable vector carrying a polynucleotide encoding a target peptide is prepared in an expressible form (eg, downstream of a regulatory sequence corresponding to a promoter sequence) and transformed into an appropriate host cell. The host cell is then cultured to produce the peptide of interest. The peptide can also be produced in vitro using an in vitro translation system.

IV. Polynucleotides

The invention provides polynucleotides encoding any of the foregoing peptides of the invention. These include polynucleotides derived from the naturally occurring Foxp3 gene, and their conservatively modified nucleotide sequences. As used herein, the phrase "conservatively modified nucleotide sequence" refers to a sequence that encodes the same or substantially the same amino acid sequence. Because of the degeneracy of the genetic code, there are many functionally identical nucleic acids encoded as any given protein. For example, the codons GCA, GCC, GCG, and GCU are all encoded as amino acid alanine. Thus, at each position where the codon is assigned to alanine, the codon can be altered to any corresponding codon without altering the encoded multi-peptide. Nucleic acid variation For "silent variation", it is a conservatively modified variation. Each of the nucleic acid sequences encoded herein as a peptide also describes each of the possible silent variations of the nucleic acid. Those skilled in the art will be aware of each codon in a nucleic acid (except for AUG, which is typically the only codon for methionine, which is typically the only codon for tryptophan) and can be modified to functionally The same molecule. In summary, each silent variation of a nucleic acid encoded as a peptide is implicitly recited in each sequence.

The polynucleotide of the present invention may be composed of DNA, RNA and derivatives thereof. The DNA is suitably composed of a base such as A, T, C and G. For RNA, T is replaced by U.

The Foxp3 polynucleotide of the invention is encoded as a plurality of Foxp3 peptides of the invention with or without intervening amino acid sequences. For example, the intervening amino acid sequence can provide the polynucleotide or the translated peptide to an open site (eg, an enzyme recognition sequence). Furthermore, the polynucleotide may include any additional sequences in addition to the coding sequence encoding the peptide of the present invention. For example, the polynucleotide can be a recombinant polynucleotide, including regulatory sequences necessary for expression of the multi-peptide. In general, such recombinant polynucleotides can be prepared by manipulation of polynucleotides using conventional recombinant techniques, using, for example, polymerases and endonucleases.

Both recombinant and chemical synthesis techniques can be used to produce the polynucleotides of the invention. For example, the polynucleotide can be produced by insertion into a suitable vector that can be expressed when transfected into a competent cell. Alternatively, the polynucleotide can be amplified or expressed in a suitable host using PCR techniques (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring). Harbor Laboratory, New York, 1989). Alternatively, the polynucleotide can be synthesized using a solid phase technique as described by Beaucage S. L. & Iyer R. P., Tetrahedron 48: 2223-311, 1992; Matthes et al., EMBO J 3: 801-5, 1984.

V. Pharmaceutical preparations

Since Foxp3 has been identified as a molecule that regulates T(reg-T) cells, which act to maintain constant immunity, the Foxp3 peptide of the present invention or the polynucleotide encoding the Foxp3 peptide can be used to modulate T-reg cells. Accordingly, the present invention provides a pharmaceutical preparation for regulating T-reg cells comprising one or more peptides of the present invention or a polynucleotide encoding the peptide as an active ingredient.

Here, "modulating" T-reg cells represents modifying the state of T-reg cells in vivo, for example, inhibiting T-reg cell proliferation or suppressing its function. T-reg cells are thought to be one of the main roles in inhibiting various types of immune responses, and "repressing T-reg cell function" herein means reducing the ability of T-reg cells to suppress an immune response. In particular, the peripheral action of T-reg cells is called peripheral tolerance (Miescher S et al., J Immunol 136: 1899-907, 1986; Young RC et al., Am J Med 52: 63-72). , 1972; Alexander JP et al., Cancer Res 53: 1380-7, 1997; Horiguchi S et al., Cancer Res 59: 2950-6, 1999; Kono K et al., Clin Cancer Res 11: 1825-8, 1996; Kiessling R et al., Cancer Immunol Immunother 48: 353-62, 1999; Fontenot JD et al., Nat Immunol 4: 330-6, 2003, Hori S et al., Science 299: 1057-61, 2003; Khattri R et al., Nat Immunol 4: 304-6, 2003). T-reg cells provide an immunosuppressive effect, for example, in a cancer patient. Therefore, the Foxp3 peptide of the present invention or the polynucleotide encoding the Foxp3 peptide which is excessively expressed in T-reg cells can be used as a pharmaceutical preparation for treating cancer (for example, a vaccine).

In the present invention, the phrase "vaccine" (also referred to as an immunological composition) refers to a substance which acts to induce anti-tumor immunity or to modulate the immunity of T-reg when inoculated into an animal.

The pharmaceutical preparation of the present invention can be used for the treatment and/or prevention of cancer in a body such as a human and other mammals including but not limited to: mouse, rat, guinea pig, rabbit, cat, dog, sheep , goats, pigs, cattle, horses, monkeys, baboons and chimpanzees, especially animals of economic value or domesticated animals.

According to the invention, the peptide comprising the amino acid sequence SEQ ID NO: 3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 or 74 is HLA-A24 or HLA-A02 limits the epitope determinant and induces an effective and specific immune response against T-reg cells expressing Foxp3. Therefore, the present pharmaceutical preparation is intended for administration to an individual whose HLA antigen is one of HLA-A24 or HLA-A02.

The cancer to be treated by the pharmaceutical preparation of the present invention is not limited, and includes, among them, Foxp3 exhibiting all kinds of cancers of the individual. Exemplary cancers include: breast cancer, AML, gallbladder cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffuse stomach Cancer, liver cancer, lung cancer, lymphoma, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, small cell lung cancer, soft tissue tumor, and testicular tumor.

The pharmaceutical preparation of the present invention comprising a Foxp3 peptide or a polynucleotide encoding a Foxp3 peptide may optionally include other therapeutic substances as an active ingredient as long as the substance does not inhibit T-reg cells which are concerned with the peptide. Adjust the effect. For example, the formulation may include an anti-inflammatory agent, an analgesic, a chemotherapeutic agent, and the like. In addition to including other therapeutic substances in the medicament, the pharmaceutical preparation of the present invention can be administered sequentially or simultaneously with one or more other pharmacological agents. The amount of the pharmaceutical agent and the pharmacological agent depends, for example, on the type of pharmacological agent to be used, the disease to be treated, and the time course and route of administration.

It will be appreciated that in addition to the ingredients specifically mentioned herein, the pharmaceutical preparations of the present invention may include other agents conventional in the art related to this type of formulation.

In one embodiment of the invention, the pharmaceutical preparations can be included in the articles of manufacture and in kits containing therapeutic conditions for the treatment of a condition to be treated, such as cancer. The article of manufacture may comprise: a container containing the pharmaceutical preparation and labeled. Suitable containers include bottles, small glass vials, and test tubes. The container can be formed from a variety of materials such as glass or plastic. The label on the container should indicate that the agent is used to treat or prevent more than one condition of the disease. The label can also display a description of the investment, and the like.

In addition to the above containers, the kit comprising the pharmaceutical preparation of the present invention may optionally include a second container containing a pharmaceutically acceptable diluent. May include other materials that will be needed from a business or user perspective, including other Granules, thinners, filters, needles, syringes and instructions for use.

The pharmaceutical composition may be in the form of a pack or a dispensing device, including one or more unit dosage forms containing the active ingredient. The bag may for example comprise a metal or plastic foil, such as a blister. The package or dispensing device can be attached to the instructions.

(1) A pharmaceutical preparation containing the peptide as an active ingredient The peptide of the present invention can be directly administered as a pharmaceutical preparation, and can be formulated by a conventional formulation method as needed. In this case, in addition to the peptide of the present invention, an additional carrier, an excipient or the like which is usually used for a drug may be appropriately included without particular limitation. Examples of such a carrier include sterile water, physiological saline solution, phosphate buffer solution, and culture solution. Furthermore, the pharmaceutical preparation may optionally include a stabilizer, a suspension, a preservative, a surfactant, and the like. The pharmaceutical preparation of the present invention can be used for the treatment and/or prevention of cancer, especially for regulating T-reg cells.

The peptide of the present invention can be combined to form two or more of the Foxp3 peptide of the present invention to induce CTL in vivo. The Foxp3 peptides can be combined with each other either by mixing or using standard techniques. For example, the Foxp3 peptide can be expressed as a single multi-peptide sequence. The peptides in the combination may be the same or different. By administering the Foxp3 peptide of the present invention, the peptide is present at a high density on the HLA antigen of the antigen-presenting cell, and then inducing a CTL that specifically reacts with the complex formed between the presented peptide and HLA antigen reaction. . Alternatively, an antigen-presenting cell immobilized with a Foxp3 peptide of the present invention on a cell surface can be obtained by removing dendritic cells from an individual stimulated with the peptide of the present invention by re-administering the Foxp3 The peptide-loaded dendritic cells give the individual an induction of CTL in the individual and the result is for the target Invasiveness of cells can be increased.

A pharmaceutical preparation for modulating T-reg cells comprising the Foxp3 peptide of the present invention as an active ingredient, optionally containing an adjuvant for efficiently establishing cellular immunity, or the same can be administered together with other active ingredients, and It can be formulated into granules and then administered. An adjuvant is a compound that, when administered together with an immunologically active protein (or sequentially), enhances the immune response against the protein. Applicable adjuvants include those described in the literature (Clin Microbiol Rev 7: 277-89, 1994). Exemplary adjuvants include, but are not limited to, aluminum phosphate, aluminum hydroxide, alum, cholera toxin, salmonella toxin, and the like.

Furthermore, it is convenient to use a liposome formulation in which the Foxp3 peptide is bound to a particle formulation of several-mcm diameter beads, and the lipid is bound to the peptide formulation.

Some embodiments of the pharmaceutical preparations of the invention comprise a component that primes a cytotoxic T lymphocyte. Lipids have been identified as substances that enable CTLs to initialize against viral antigens in vivo. For example, a palmitic acid residue may be attached to the epsilon- and a-amino group of the amine acid residue and linked to the peptide of the present invention. The lipidated peptide can then be administered directly to the granules or microparticles, encapsulated in the liposomes or emulsified in an adjuvant. As a further lipid-initiating CTL reaction, E. coli lipoproteins, such as triptolide-S-glycerylcysteine-serine-serine (P3CSS), can be covalently bonded to the appropriate peptide to initialize the CTL. (See, for example, Deres et al., Nature 342: 561, 1989).

The administration method may be oral, intradermal, subcutaneous, intravenous injection, etc., and Systemic administration or topical administration to the vicinity of the target site is useful. The administration can be carried out by a single administration or by additional injection. The dose of the peptide of the present invention can be appropriately adjusted depending on the disease to be treated, the age of the patient, the body weight, the administration method, and the like, and is usually 0.001 mg to 1000 mg, for example, 0.001 mg to 1000 mg, for example, 0.1 mg to 10 mg, and each number It is given every day until every few months. Those skilled in the art can appropriately select the appropriate dosage.

(2) A pharmaceutical preparation comprising a polynucleotide as an active ingredient The pharmaceutical preparation of the present invention may comprise a nucleic acid which is encoded in the expressible form as the Foxp3 peptide disclosed herein. Here, the phrase "presentable form" means that when the polynucleotide is introduced into a cell, it exhibits a multi-peptide in vivo and induces anti-tumor immunity. In one embodiment, the nucleic acid sequence of the polynucleotide of interest comprises an regulatory element necessary for the expression of the polynucleotide in the target cell. The polynucleotide can be stably inserted into the genome of the target cell (see, for example, Thomas KR & Capecchi MR, Cell 51: 503-12, 1987 for a description of homologous recombination cassette vectors). See, for example, Wolff et al., Science 247: 1465-8, 1990; US Patent Nos. 5, 580, 859; 5, 589, 466; 5, 804, 566; 5, 739, 118; 5, 736, 524; 5, 679, 647; and WO 98/04720. DNA-based delivery technologies include "naked DNA", promoting (bupivicaine, polymer, peptide mediator) delivery, cationic lipid complexes, and delivery of particulate media ("gene guns") or pressure media (see, for example, US patent numbers) 5,922,687).

The peptide of the present invention can also be expressed by a viral or bacterial carrier. Examples of performance vectors include attenuated viral hosts such as vaccinia or fowlpox viruses. this The method involves the use of a vaccinia virus, for example as a vector to express a nucleotide sequence encoded as the peptide. Upon introduction into the host, the recombinant vaccinia virus exhibits the immunogenic peptide and elicits an immune response. Vaccinia vectors and methods for use in immunological assay procedures are described, for example, in U.S. Patent No. 4,722,848. The other vector is BCG (Bacille Calmette Guerin). The BCG vector is described in Stover et al., Nature 351: 456-60, 1991. Many other vectors are useful for therapeutic administration or immunization, such as adenovirus and adeno-associated viral vectors, retroviral vectors, Salmonella typhimurium vectors, detoxified anthrax toxin vectors, and the like. See, for example, Shata et al., Mol Med Today 6: 66-71, 2000; Shedlock et al. J Leukoc Biol 68: 793-806, 2000; Hipp et al., In Vivo 14: 571-85, 2000.

Delivery of the polynucleotide to the patient can be direct, in which case the patient is directly exposed to the vector carrying the polynucleotide, or indirectly, in which case the cell is first transferred to the polynucleotide of interest. Shape, then transplanted to the patient. These two methods are known and are referred to as in vivo or ex vivo gene therapy.

For general comments on gene therapy methods, see Goldspiel et al., Clinical Pharmacy 12: 488-505, 1993; Wu and Wu, Biotherapy 3: 87-95, 1991; Tolstoshev, Ann Rev Pharmacol Toxicol 33: 573-96, 1993; Mulligan, Science 260: 926-32, 1993; Morgan & Anderson, Ann Rev Biochem 62: 191-217, 1993; Trends in Biotechnology 11(5): 155-215, 1993). Methods commonly known in the Art of recombinant DNA technology which can be used are described in eds. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY, 1993; and Krieger, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY , 1990.

The administration method may be oral, intradermal, subcutaneous, intravenous injection or the like, and systemic administration or topical administration to the vicinity of the target site is useful. The administration can be carried out by a single administration or by additional injection. The dose of the polynucleotide in the appropriate carrier or the polynucleotide which is encoded as the polynucleotide of the peptide of the present invention can be appropriately adjusted according to the disease to be treated, the age of the patient, the body weight, the administration method, and the like. It is usually from 0.001 mg to 1000 mg, for example from 0.001 mg to 1000 mg, for example from 0.1 mg to 10 mg, and can be administered once every few days to every few months. Those skilled in the art can appropriately select the appropriate dosage.

(3) Exogenous body Alternatively, the invention provides an intracellular vesicle called an exosome that presents a complex formed by the peptide of the invention and the HLA antigen on the surface. The exosome can be prepared, for example, by using the method described in Japanese Patent Publication No. Hei 11-510507 and Japanese Patent Publication No. Hei. No. Hei. The exosome of the present invention can be vaccinated, similar to the peptide of the present invention.

The type of HLA antigen used must be consistent with the type of HLA antigen of the individual in need of treatment and/or prevention. For example, for the Japanese, HLA-A24, Especially HLA-A2402, is often appropriate.

Regarding the HLA antigen, the use of the A-24 or A-02 type which is highly expressed in Japanese and Caucasian is advantageous for obtaining an effective result, and it is useful to use a subtype including A-2402 and A-0201. In general, the HLA antigen type of a patient in need of treatment is first examined clinically, so that a peptide having a high level of binding affinity for the antigen or a peptide exhibiting cytotoxic T cell (CTL) ability with an antigen can be appropriately selected. Furthermore, in order to obtain a peptide exhibiting high binding affinity and CTL inducing ability, one, two or several amino acids may be substituted or added depending on the amino acid sequence of the naturally occurring Foxp3 partial peptide.

(4) antigen presenting cells The present invention also provides antigen presenting cells (APCs) which exhibit a complex formed by an HLA antigen and a peptide of the present invention on a surface. The APCs obtained by contacting the peptide of the present invention or the nucleotide encoding the peptide of the present invention can be prepared from individuals who are targets for treatment and/or prevention, and can be administered to the individuals. Or as a vaccine in combination with other drugs or cytotoxic T cells comprising the peptide of the present invention, exosome.

The APCs are not limited to any kind of cells, and include dendritic cells (DC), Langerhans cells, macrophages, B cells, and activated T cells, all of which are known to exhibit protein antigens on their cell surface. To make the lymphocytes know. Since DC is a representative APC having the strongest CTL induction in APC, DC is particularly useful as the APC of the present invention.

For example, APC can induce dendritic cells by mononuclear cells from peripheral blood, These are obtained by contacting (stimulating) the peptide of the present invention in vitro, ex vivo or in vivo. When the peptide of the present invention is administered to the individual, the APC immobilized with the peptide of the present invention is induced in the individual. "Inducing APC" includes contacting (stimulating) a cell with the peptide of the present invention or a nucleotide encoding the peptide of the present invention to present a complex formed by the HLA antigen and the peptide of the present invention on the cell surface. on. Alternatively, after immobilizing the peptide of the present invention to APC, APC can be administered to the individual as a vaccine. For example, ex vivo administration can comprise the steps of: a: collecting APC from an individual, and b: contacting the APC of step a with the peptide.

The APC obtained from step b can be administered to the individual as a vaccine.

According to the state of the invention, APC has a high level of CTL inducing ability. This APC having a high level of cytotoxic T cell inducing ability can be prepared by a method comprising the step of transferring a gene comprising a polynucleotide encoding the peptide of the present invention to APC in vitro. The gene to be introduced may be DNA or RNA. The method to be introduced is not particularly limited, and methods conventionally known in the art, such as lipofection, electroporation, and calcium phosphate method, can be used. More specifically, Cancer Res 56 can be performed according to the methods described below: 5672-7, 1996; J Immunol 161: 5607-13, 1998; J Exp Med 184: 465-72, 1996; International Publication 2000-509281 Translation version. By transferring the gene into APC, the gene undergoes transcription, translation, etc. in the cell, and the resulting protein is then treated with MHC class I or II and via a presentation path. Present some peptides.

(5) Cytotoxic T cells Cytotoxic T cells that are induced to combat any of the Foxp3 peptides of the present invention are believed to potentiate the targeting of the immune system to T-reg cells in vivo and thus can be used as a vaccine similar to the peptide. Accordingly, the invention provides isolated cytotoxic T cells that are induced via any of the peptides of the invention.

Such cytotoxic T cells can be obtained by (1) administering a peptide of the present invention to one body, or (2) contacting (stimulating) the peptide of the present invention with APC and CD8-derived in vitro. Positive cells or peripheral blood mononuclear white blood cells.

The cytotoxic T cell, which is induced by APC which exhibits the peptide of the present invention, can be derived from an individual who is a target for treatment and/or prevention, and can be administered by itself, or for adjustment effect. For the purpose, it is administered together with other drugs including the peptide or exosome of the present invention. The resulting cytotoxic T cells specifically act on the target cells presenting the peptide of the invention, for example, for the same peptide to be induced. The target cell may be a cell which is endogenous to express Foxp3, or a cell transfected with the Foxp3 gene, and a cell which exhibits the peptide of the present invention on the cell surface due to stimulation with such a peptide, and may also be a target of attack. .

(6) TCRs The present invention also provides a composition comprising a nucleic acid encoding a multi-peptide, which is capable of forming a secondary unit of a T cell receptor (TCR), and a method of using the composition. The TCR subunit has the ability to form a TCR, the TCR There is specificity for T cells given to cells exhibiting the peptide of the present invention. By using techniques known in the art, α- and β-strand nucleic acids of TCR subunits of CTLs induced by one or more of the peptides of the present invention can be identified (WO2007/032255 and Morgan et al., J). Immunol, 171, 3288 (2003)). The derivatized TCR preferably binds to the target cell exhibiting the Foxp3 peptide with high affinity, and optionally kills the target cell exhibiting the Foxp3 peptide in vivo and in vitro.

A nucleic acid encoding a TCR subunit can be introduced into a suitable vector, such as a retroviral vector. Such vectors are well known in the art. The nucleic acid or a useful vector comprising the nucleic acid can be transferred into a T cell, preferably from a patient. Advantageously, the present invention provides an off-the-shelf composition that allows rapid modification of a patient's own T cells (or other mammalian T cells) to rapidly and easily produce modified T cells. The modified T cell has superior T-reg cell killing properties.

Further, the present invention provides a CTL which is produced by transduction of a nucleic acid encoding a TCR subunit polypeptide which binds to the Foxp3 peptide of the present invention. The transduced CTL is capable of forming T-reg cells in vivo and is cultured in vitro by well-known culture methods (for example, Kawakami et al., J Immunol., 142, 3452-3461 (1989)). The T cells of the present invention are useful for forming an immunological composition for treating or preventing cancer in a patient in need of treatment or protection (WO2006/031221).

VI. Method of using Foxp3 peptide

The Foxp3 peptide of the present invention and the polynucleoside encoded by the Foxp3 peptide Acid, which can be used to induce APC and CTL. The Foxp3 peptide and polynucleotide can be used in combination with other compounds as long as the compounds do not inhibit their CTL inducing ability. Therefore, any of the above pharmaceutical preparations of the present invention can be used in the following methods.

(1) Method for inducing antigen presenting cells (APCs) Accordingly, the present invention provides a method of using the peptide of the present invention or a polynucleotide encoding the peptide to induce APC. Induction of APC can be carried out as described in the item "V-(4) antigen presenting cells". The present invention also provides a method for inducing APC having a high level of cytotoxic T cell inducing ability, the induction of which is described above as "V-(4) antigen presenting cells". Alternatively, according to the present invention, the use of a peptide selected from the group consisting of amino acid sequence SEQ ID NO: 3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 or 74 is provided. A Foxp3 peptide, or a polynucleotide encoding the Foxp3 peptide, is used to make a pharmaceutical composition comprising one of antigen presenting cells. Furthermore, the present invention also provides a peptide selected from the amino acid sequence of SEQ ID NO: 3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 or 74. A Foxp3 peptide, or a polynucleotide encoding the Foxp3 peptide, for inducing antigen to present cells.

(2) Method of Inducing Cytotoxic T Cells Further, the present invention provides a method of inducing CTL by using the Foxp3 peptide of the present invention or a polynucleotide encoding the Foxp3 peptide. When the Foxp3 peptide of the present invention is administered to a single body, CTL is induced in the individual and the strength of the immune system targeted to the T-reg cells is enhanced. Alternatively, the invention may be used in an ex vivo treatment method in which an individual-derived APC and CD8-positive cells or peripheral blood mononuclear leukocytes are contacted (stimulated) with the peptide of the present invention in vitro, after inducing CTL, The cells are returned to the individual. For example, the method can comprise the steps of: a: collecting APC from an individual, b: contacting the APC of step a with the peptide, c: mixing the APC of step b with CD8 ⁺ T cells, and coculturing for induction of cytotoxicity T cells, and d: CD8 ⁺ T cells were harvested from the co-culture of step c.

The cytotoxic activity of CD8 ⁺ T cells obtained from step d can be administered to the individual as a vaccine. The APC to be mixed with CD8 ⁺ T cells in the above step c can also be prepared by transferring the gene encoding the peptide of the present invention into APC, and the details are detailed in "V-(4) antigen presenting cells"; Without being limited thereto, any APC or exosome that effectively presents the peptide of the present invention to T cells can be used in the present method. Or, in accordance with the present invention. Providing a Foxp3 peptide selected from a peptide comprising the amino acid sequence SEQ ID NO: 3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 or 74, or A polynucleotide encoding the Foxp3 peptide is used to make a pharmaceutical composition comprising CTL. Again. The present invention further provides the Foxp3 which is selected from the peptide comprising the amino acid sequence SEQ ID NO: 3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 or 74. A peptide, or a polynucleotide encoding the Foxp3 peptide, for inducing CTL.

(3) Regulating immunosuppression As described above, the peptide, polynucleotide, exosome, APC and CTL of the present invention can be used as a vaccine to modulate (i.e., inhibit) T-reg cells. Because T-reg is thought to inhibit multiple types of immune responses, especially CTL cytotoxicity One of the main roles of sexual activity, the ability of the peptides, polynucleotides, exosomes, APCs, and CTLs of the present invention, can also be used to counteract immunosuppression, particularly CTL cytotoxic activity. In summary, the present invention provides a method of modulating T-reg cells and a method of modulating (ie, offsetting) immunosuppression comprising administering to a subject in need thereof a peptide, a polynucleotide, an exosome, Steps for APC or CTL. Furthermore, the present invention also provides for the use of peptides selected from the group consisting of amino acid sequences SEQ ID NO: 3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 or 74. A Foxp3 peptide, or a polynucleotide encoding the Foxp3 peptide, to produce an immunological composition for modulating immunosuppression. Alternatively, the present invention relates to Foxp3 selected from peptides comprising the amino acid sequence SEQ ID NO: 3-5, 7-9, 12, 15-19, 22, 24, 27-30, 37, 67 or 74. A peptide, or a polynucleotide encoding the Foxp3 peptide, is used to modulate immunosuppression.

Here, modulation of immunosuppression represents administration of a peptide, polynucleotide, exosome, APC or CTL of the invention, resulting in any kind of change in vivo. In some embodiments, the alterations caused by the peptides, polynucleotides, exosomes, APCs, and CTLs of the invention are those that reduce the level of immunosuppression (inhibition or offsetting immunosuppression), i.e., induce anti-immunosuppression. Accordingly, the present invention further provides a method of inducing anti-immunosupsis comprising the step of administering to a subject in need thereof a peptide, polynucleotide, exosome, APC or CTL of the invention.

In general, anti-immunosuppression includes an immune response as follows: - induction of cytotoxic lymphocytes against T-reg expressing Foxp3, - induction of antibodies recognizing T-reg of Foxp3, and - Inducing anti-Treg cytokine production.

Therefore, when a certain protein is inoculated into an animal, any immune reaction is induced, and the protein is judged to have an anti-immunosuppressive induction effect. Anti-immunosuppression induced by a protein can be detected by observing the host immune system against the in vivo or in vitro response of the protein.

For example, methods for detecting induced (i.e., activating) cytotoxic T lymphocytes are well known. In particular, it is known that foreign substances entering a living body are presented to T cells and B cells by the action of antigen presenting cells (APC). T cells that respond to the antigen presented by APC are differentiated into cytotoxic T cells (or cytotoxic T lymphocytes; CTL) by antigen-specific stimulation, and then proliferate (this is called activated T cells). Thus, a CTL induced by a peptide can be evaluated by presenting the peptide to T cells with APC and detecting CTLs for induction (ie, proliferation, IFN-γ production, and cytotoxic activity). Furthermore, APC has the function of activating CD4+ T cells, CD8+ T cells, macrophages, eosinophils, and NK cells. Since CD4+ T cells are also important in anti-tumor immunity, the anti-tumor immunity induction effect of the peptide can be used as an index for evaluating the activation effect of such cells.

The use of dendritic cells (DCs) as APCs to assess the effects of inducing CTLs is well known in the art. According to this method, a test peptide is first contacted with DC and then the DC is contacted with T cells. Upon exposure to DC, T cells that are cytotoxic against cells expressing the peptide of interest (i.e., present on HLA molecules) are detected, indicating that the test peptide has activity to induce cytotoxic T cells. The activity of CTL against T-reg can be detected using, for example, ⁵¹ Cr-calibrated tumor cell lysis as an indicator. Alternatively, methods for assessing T-reg damage using ³ H-thymidine uptake activity or LDH (lactose dehydrogenase)-release as indicators are also well known and can be used in the present invention.

In addition to DC, peripheral blood mononuclear cells (PBMCs) can also be used as APC. Induction of CTL It is reported that the cultivation of PBMC in the presence of GM-CSF and IL-4 is promoted. Similarly, CTL has been shown to culture PBMC in the presence of keyhole limpet hemocyanin (KLH) and IL-7, which is induced.

The test peptide having CTL-inducing activity has been confirmed by such methods, and is a peptide having DC activation, and is accompanied by CTL-inducing activity. Therefore, the Foxp3 peptide which induces CTL against tumor cells is useful as a vaccine against T-reg. Furthermore, APC which induces CTL anti-T-reg ability by contact with the Foxp3 peptide is also useful as a vaccine against T-reg. Furthermore, a cytotoxic CTL can be obtained by presenting the peptide antigen in APC, and can also be used as a vaccine against T-reg. Such a method of regulating the immunity against T-reg using APC and CTL is called cell immunotherapy and is included in the present invention.

In general, when multi-peptide is used in cellular immunotherapy, the efficiency of CTL induction is known to increase due to the combination of most peptides with different structures and the contact with DC. Therefore, when DCs are stimulated with protein fragments, it is advantageous to use a mixture of multiple types of fragments.

Alternatively, induction of anti-immunosuppression by a peptide can be confirmed by observing production of an antibody that induces anti-T-reg. For example, when one of the individuals immunized with the peptide, such as a human patient, a laboratory animal, induces a peptide against An antibody, and when T-reg cells are inhibited by such antibodies, the peptide is judged to have an ability to induce anti-immunosuppression.

Anti-immunosuppression is induced by administration of the vaccine of the present invention, and this induction can disrupt immunosuppression. This effect can be statistically significant. For example, at an observation level, the level of significance is 5% or less, wherein the regulation of the vaccine against T-reg is compared with the control group not administered with the vaccine. For example, Student's t-test, Mann-Whitney U-test or ANOVA can be used for statistical analysis.

When APC or CTL is used as the vaccine of the present invention, T-reg can be adjusted (i.e., inhibited) by, for example, an ex vivo method. More specifically, the PBMC of the individual receiving treatment or prevention is collected, the cells are contacted ex vivo with the multi-peptide, and after inducing APC or CTL, the cells can be administered to the individual. APC can be introduced into PBMC ex vivo by a vector encoding a multi-peptide. APC or CTL induced in vitro can be colonized in vitro prior to administration. Cellular immunotherapy can be performed more efficiently by colonizing and culturing cells that have high activity in the target cells. Furthermore, APCs and CTLs isolated in this manner can be used for cellular immunotherapy, not only for individuals who derive the cells, but also for similar disease types in other individuals.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Although the invention may be practiced or tested using methods and materials similar or equivalent to those described herein, suitable methods and materials are described below. All publications, patent applications, and other references cited herein are hereby incorporated by reference in their entirety in the entirety The specification of the present invention shall prevail. In addition, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

The embodiments are presented below to illustrate the invention and to assist those skilled in the art to make and use. These examples are in no way intended to limit the scope of the invention.

Example Materials and methods

Cell lines A24LCL cells (HLA-A24/24), T2 cells (HLA-A02/02), human B-lymphoblasts, 293T and COS7 were purchased from ATCC.

The candidate peptide candidate derived from Foxp3 is combined with the predicted software " BIMAS " (http://bimas.dcrt.nih.gov/cgi-bin/molbio/ken_par ker_comboform ), predicted to be associated with HLA-A*2402 and HLA- The A*0201 molecule binds to the 9-mer and 10-mer peptides derived from Foxp3, and the algorithm is based on Parker KC, et al. ((1994) J Immunol.; 152(1): 163-75.) and Kuzushima K. , et al. ((2001) Blood.; 98(6): 1872-81.). These peptides were synthesized by Sigma (Sapporo, Japan) according to standard solid phase synthesis and purified by reverse phase HPLC. Purity (>90%) and the identity of the peptide were determined by analytical HPLC and mass spectrometry. The peptide was dissolved in dimethyl hydrazine (DMSO) at a concentration of 20 mg/ml and stored at -80 °C.

In vitro CTL induction Mononuclear-derived dendritic cells (DCs) were used as antigen-presenting cells (APCs) to induce CTL responses against peptides present on HLA. DCs are produced in vitro in an alternative manner (Horiguchi S. et al. Cancer Res. 59: 2950-6). Specifically, peripheral blood mononuclear cells (PBMC) isolated from normal volunteers (HLA-A*2402 and/or HLA-A*0201) in a Ficoll-Plaque (Pharmacia) solution are attached to a plastic Petri dish. (Becton Dickinson) is separated to make it to the mononuclear sphere portion. Mononuclear spheres were enriched in 1000 U/ml GM-CSF (R&D System) and 1000 U/ml IL-4 (R&D Systems) and cultured in AIM-containing 2% heat-inactivated serum (AS) obtained from itself. V medium (Invitrogen). After 7 days of culture, the cytokine-produced DC was pulsed at 20 mcg/ml for 4 hours in the presence of β2-microglobulin at 3 mcg/ml in AIM-V medium at 20 °C. The DCs of these peptides were then inactivated with mitomycin C (MMC) (30 mcg/ml, 30 min) and mixed with CD8 ⁺ T cells obtained in their own ratio of 1:20, CD8 ⁺ T cells. The CD8 positive segregation kit (Dynal) was used for positive selection. These cultures were placed on a 48-well plate (Corning); each well contained 1.5x10 ⁴ peptide-pulsed DCs, 3x10 ⁵ CD8 ⁺ T cells, and 10 ng/ml in AIM-V/2% AS 0.5 ml. IL-7 (R&D System). After 3 days, the cultures were supplemented with IL-2 (CHIRON) to a final concentration of 20 IU/ml. On days 7 and 14, the cells were stimulated again with the DC obtained by the peptide pulse itself. DCs were prepared each time in the same procedure as above. After the peptide stimulation of the third round on the 21st day, the CTL activity against the peptide pulsed A24LCL cells or T2 cells was tested.

The procedure for expanding the CTL allows CTL to be used in culture similar to Riddell, et al. (Walter et al., N Engl J Med 333(16): 1038-44, 1995; Riddell et al., Nat Med 2(2) : 216-23, 1996 Feb) The method described expands the population. A total of 5× ^{10 4} CTL and 2 human B-lymocyte strains were resuspended in AIM-V/5% AS 25 ml, and inactivated with MMC in a 40 ng/ml anti-CD3 monoclonal antibody (Pharmingen). After 1 day of culture, 120 IU/ml of IL-2 was added to the culture. On days 5, 8 and 11, fresh AIM-V/5% AS containing 30 IU/ml IL-2 was added to the culture.

CTL-specific activity To detect the specificity of CTL, the IFN-γ ELISPOT assay and the IFN-γ ELISA were used. Briefly, peptides pulsed with A24-LCL, T2 cells (1x10 ⁴ /well) or cells endogenously expressing Foxp3 and HLA molecules were prepared as stimulator cells. A CTL cell line cultured in well 48 was used as a responsive daughter cell. The IFN-γ ELISPOT assay and the IFN-γ ELISA were performed according to the manufacturing procedure.

Immunogenicity of the epitope-determining peptide in BALB/c mice To prime the peptide-specific CTL, each mouse was immunized with a 100 mlcl vaccine mixture containing 50 ml of HLA-A24-restricted peptide and 50 ml IFA. The vaccine was injected subcutaneously into the right flank of the mouse as the first immunization on the 0th day, and injected into the left flank as the second immunization on the 7th day. On day 14, spleen cells from vaccinated mice were used as responsive cells, and RLmalel cells pulsed with or without peptides were used as stimulator cells for the IFN-γ ELISPOT assay.

In vivo anti-tumor effect 4T1 cells (1×10 ⁵ per mouse) were injected subcutaneously on day 0 into the right flank of BALB/c mice. On days 3 and 10, hFoxp3-252 (KLSAMQAHL: SEQ ID NO: 17) or mFoxp3-252 (KLGAMQAHL: SEQ ID NO: 88) IFA-conjugated peptide was used for inoculation.

Analysis of the affinity of the Foxp3-9-252 substitute for HLA molecules was performed in an IFN-[gamma] ELISA assay to test the affinity of the substituted peptide for the HLA-A2 molecule. CTLs induced by Foxp3-9-252-WT (KLSAMQAHL: SEQ ID NO: 17) were used as responsive daughter cells, and by using Foxp3-9-252-WT, Foxp3-9-252-9V (KLSAMQAHV: SEQ ID NO: 95) and HIV-A02 (SLYNTYATL) peptide were incubated with 37 degrees Celsius for 2 hours to prepare T2 cells as stimulator cells. Each peptide was subjected to a peptide pulse for T2 cells at a wide range of concentrations (10-10 ^-4 mcg/ml).

result

Prediction of HLA-A24 and HLA-A2 binding peptides derived from Foxp3 Tables 1, 2 and 3 show the HLA-A*2402 binding peptide or HLA-A*0201 binding peptide of Foxp3 protein in the order of high binding affinity prediction fraction. A total of 60 peptides were selected with the possibility of HLA-A24 binding activity, and 26 peptides had the possibility of HLA-A2 binding activity.

The starting position represents the amino acid number from the N-terminus of Foxp3. The combined scores are from those described in the Materials and Methods section of the "BIMAS".

The starting position represents the amino acid number from the N-terminus of Foxp3. The combined scores are from those described in the Materials and Methods section of the "BIMAS".

The starting position represents the amino acid number from the N-terminus of Foxp3. The combined scores are from those described in the Materials and Methods section of the "BIMAS".

Stimulation of T cells using predicted peptides restricted by HLA-A2402 CTLs against peptides derived from Foxp3 protein were generated according to the method of the "Materials and Methods". The CTL obtained, as shown by the IFN-γ ELISPOT assay, showed specific CTL activity as shown in Figures 1A and 1B. In Figure 1A, cells stimulated with Foxp3-A24-9-363 in Yujing #2 and 7, cells stimulated with Foxp3-A24-9-366 in #1 and #6, and #5 via Foxp3-A24-9- 190-stimulated cells, #7, cells stimulated with Foxp3-A24-10-87 and Foxp3-A24-10-60 showed potent IFN-γ production compared to the control group. In Figure 1B, cells stimulated with Foxp3-A24-9-207 in Yujing #4, cells stimulated with Foxp3-A24-9-332 in #6, and cells stimulated with Foxp3-A24-9-337 in #6 And the cells stimulated with #pxp3-A24-10-114 in #1 showed an effective IFN-γ production compared to the control group.

Stimulation of T cells using predicted peptides restricted by HLA-A0201 The CTLs showing detectable specific CTL activity in the IFN-γ ELISPOT assay are shown in panels 2A, 2B and 2C. In Figure 2A, cells stimulated with Foxp3-A2-9-390 in Yujing #2, cells stimulated with Foxp3-A2-9-69 in #2, cells stimulated with Foxp3-A2-9-252 in #6, #4 via Foxp3-A2-10-359 stimulated cells, #7 via Foxp3-A2-263 stimulated cells, and #2 and #5 via Foxp3-A2-10-94 stimulated cells, compared to the control group, Shows potent IFN-γ production. In Figure 2B, cells from all wells were stimulated with Foxp3-A2-10-233, wells #6 and #7 were stimulated with Foxp3-A2-10-152, and #5 was stimulated with Foxp3-A2-10-77. Cells, cells stimulated with Foxp3-A2-10-246 and Foxp3-A2-10-94, showed potent IFN-γ production compared to the control group. In Figure 2C, cells stimulated by Foxp3-A2-9-390 in well numbers #1, 2, 4, 5, 7, 9, 11 and 12, in well numbers #5 and #11 via Foxp3-A2-9 -304 stimulated cells, cells stimulated with Foxp3-A2-9-68 in well number #7, and cells stimulated with Foxp3-A2-9-252 in well number #12, showing effective energy compared to the control group IFN-γ production.

Positive wells will establish CTL cell lines to expand the population and the implementation of IFN-γ ELISA from Foxp3-specific peptide. In the 3A, B, and C panels, CTL cell lines stimulated with Foxp3-A02-9-390 (SEQ ID NO: 15) showed potent IFN-γ production compared to the control group. In the 3D panel, CTL cell lines stimulated with Foxp3-A02-9-252 (SEQ ID NO: 17) showed potent IFN-γ production compared to the control group. In Figure 3E, CTL cell lines stimulated with Foxp3-A24-10-60 (SEQ ID NO: 75) showed potent IFN-γ production compared to the control group. In Figure 3F, CTL cell lines stimulated with Foxp3-A02-10-94 (SEQ ID NO: 27) showed potent IFN-γ production compared to the control group. In the 3G map, CTL cell lines stimulated with Foxp3-A24-10-87 (SEQ ID NO: 68) showed potent IFN-γ production compared to the control group.

Specific CTL activity against endogenously expressed Foxp3 and HLA-A*2402 or HLA-A*0201 target cells For the established CTL colonies against these peptides, examine their understanding of endogenous performance of Foxp3 and HLA - The ability of A*24 or 02 to target cells. For 293T specific CTL activity was tested, the 293T was transfected with both Foxp3 gene and HLA-A*24 or 02 full-length, which is endogenous to the specificity of Foxp3 and HLA-A*24 or 02 target cells. Model, CTL cell line produced by Foxp3-A02-9-390 (SEQ ID NO: 15) and Foxp3-A02-9-252 (SEQ ID NO: 17) was used as an effector cell for the 293T specific CTL activity. test. CTL cell lines produced by Foxp3-A02-9-390 (SEQ ID NO: 15) and Foxp3-A02-9-252 (SEQ ID NO: 17) for Foxp3 and HLA-A02 in Figures 4A and 4B Both transfected 293T showed highly specific CTL activity. In Figure 4C, a CTL cell line produced by Foxp3-A02-9-252 (SEQ ID NO: 17) showed highly specific CTL activity for 293T transfected with both Foxp3 and HLA-A24. On the other hand, it showed no significant specific CTL activity for the control group. It is clearly shown that Foxp3-A02-9-390 and Foxp3-A02-9-252 are naturally expressed on the surface of target cells bearing HLA-A02 and/or 24 molecules and recognize CTL. Furthermore, these peptides are epitope determinants and can be used in vaccines targeting T-reg expressing Foxp3.

BALB / c mice, Foxp3-A24-9-252 immunogenic peptides for the assessment to BALB / c mice, the immunogenicity of Foxp3-9-252 peptide, to each of the human Foxp3-9-252 The peptide (Foxp3-252_h; KLSAMQAHL) and the mouse Foxp3-9-252 peptide (Foxp3-252_m; KLGAMQAHL) (SEQ ID NO: 89) were immunized. After the second injection of the peptide, the peptide specific CTL activity was determined by the IFN-γ ELISPOT assay (Fig. 5).

From the spleen cells obtained from the mice injected with the peptide vaccine, effective energy IFN-γ production was detected in the well cultured with the corresponding peptide-stimulated stimulator cells, and no IFN was detected in the control well. - γ production. In Fig. 5A, Foxp3-252_h peptide specific CTL responses (M3, M4, and M5) were detected in 3 out of 5 mice, but in control mice (N1~N3) injected only with IFA. , this reaction was not detected. In Fig. 5B, Foxp3-252_m peptide specific CTL response was detected in 1 (M1) of 5 mice, but only in the control group (N1~N3) injected with IFA, this was not detected. reaction. These data show that CTLs against target cells pulsed with peptides can be induced in vivo by injection of Foxp3-252_h or Foxp3-252_m peptide vaccine.

Antitumor effect of base peptide peptide vaccine injected with Foxp3 antigen

In order to examine the anti-tumor effect with the targeted Foxp3 injected with the peptide vaccine, 4T1 tumor cells and BALB/c mice were attempted for in vivo therapeutic treatment. On day 0, 4T1 breast cancer cells were injected subcutaneously into BALB/c mice, and then vaccinated on days 3 and 10 after tumor challenge. Results Tumor growth was in the injection of Foxp3-252_h or BALB/c mice of the Foxp3-252_m peptide vaccine were significantly reduced compared to mice as a control group (Fig. 6). Considering statistical analysis, tumor growth inhibition in mice vaccinated with Foxp3 epitope determinant showed significant differences.

Amino acid substitution of Foxp3 epitope determinant peptide From the foregoing results, Foxp3-9-252 peptide (SEQ ID NO 17) was identified as an epitope peptide which was restricted by both HLA-A*2402 and HLA-A*0201. To enhance the immunogenicity of the Foxp3-9-252 peptide, a single or a group of amino acid substitutions were chosen to achieve a HLA-A*2402 or HLA-A*0201 molecule compared to the native Foxp3-9-252 peptide. Higher binding affinity; Foxp3-9-252-WT (KLSAMQAHL) (SEQ ID NO 17). The amino acid substitution binding fraction in Foxp3-9-252 (SEQ ID NO 17) was derived from the BIMAS software. Table 4 shows the binding fraction of the amino acid sequence and the substituted peptide from Foxp3-9-252 for HLA-A*2402 and 0201 molecules. The peptide binding score is derived from the BIMAS software. Six or nine substitutions, a total of 15 peptides, were predicted to have higher binding affinities for HLA-A24 or HLA-A2 molecules compared to wild type, and these peptides were synthesized (Table 4).

Next, the inventors of the present invention examined whether or not the stimulator cells using the substituted peptide pulse can be recognized by the CTL induced by Foxp3-9-252-WT peptide. As a result, the CTL induced by Foxp3-9-252-WT peptide produced IFN-γ against T2 cells pulsed with Foxp3-9-252-9V (KLSAMQAHV) (SEQ ID NO 95), and similarly, produced a confrontational IFN-γ of Foxp3-9-252-WT peptide T2 cells (Fig. 7A). Since IFN-γ production was not detected from CTL against stimulator cells without any peptide pulse, it was revealed that CTL induced by Foxp3-9-252-WT peptide was recognized to be present on HLA-A2 molecule. Foxp3-9-252-9V peptide and Foxp3-9-252-WT.

Furthermore, in order to assess whether the Foxp3-9-252-9V peptide is more affinitive to the HLA-A2 molecule than the Foxp3-9-252-WT peptide, it is used in a wide range of concentrations (10-10 ^{- 4} mcg/ml) CTL activity was examined by stimulating daughter cells pulsed with these peptides. As a result, CTLs co-cultured with each of the stimulator cells pulsed with Foxp3-9-252-WT or Foxp3-9-252-9V peptides were similar in IFN-γ production (Fig. 7B). From these data it was shown that the Foxp3-9-252-9V peptide was expressed on the HLA-A*0201 molecule, probably recognized by the CTL established with the Foxp3-9-252-WT peptide.

On the other hand, the inventors of the present invention attempted to induce CTL by using all of the substituents restricted by HLA-A*0201, including Foxp3-9-252-9V peptide. As a result, CTL was stimulated by Foxp3-9-252-3M (KLMAMQAHL) (SEQ ID NO 97), Foxp3-9-252-3L (KLLAMQAHL) (SEQ ID NO 98) or Foxp3-9-252-9V peptide And induction (Fig. 7C). Cells stimulated with Foxp3-A02-9-252-3M in wells 3 and 7 and cells stimulated with Foxp3-A02-9-252-3L in well number 7, and well numbered 8 in Foxp3-A02-9- The 252-9V stimulated cells showed peptide-dependent IFN-γ production compared to the control group. After the CTL cell line induced by Foxp3-9-252-9V stimulation was established by in vitro expansion of the population, it was determined using stimulator cells pulsed with Foxp3-9-252-WT or Foxp3-9-252-9V peptide. CTL activity. As a result, the CTL induced by Foxp3-9-252-9V stimulation recognized the stimulating cells of the pulse of Foxp3-9-252-WT peptide, and equally recognized the stimulation of the pulse of Foxp3-9-252-9V peptide. Child cells (Fig. 7D). These results strongly indicate that Foxp3-9-252-9V peptide can induce Foxp3-specific CTL, as does Foxp3-9-252-WT peptide.

Homology analysis of the antigenic peptide showed significant and specific CTL activity by the following peptide-stimulated CTLs: FOXp3-A24-9-363 (SEQ ID NO 3), FOXp3-A24-9-366 (SEQ ID NO 7) , FOXp3-A24-9-190 (SEQ ID NO 9), FOXp3-A24-9-207 (SEQ ID NO 4), FOXp3-A24-9-332 (SEQ ID NO 5), FOXp3-A24-9-337 (SEQ ID NO 8), FOXp3-A24-10-114 (SEQ ID NO 12), FOXp3-A2-9-390 (SEQ ID NO 15), FOXp3-A2-9-69 (SEQ ID NO 16), FOXp3 -A2-9-252 (SEQ ID NO 17), FOXp3-A2-10-359 (SEQ ID NO 22), FOXp3-A2-10-263 (SEQ ID NO 24), FOXp3-A2-10-94 (SEQ ID NO 27), FOXp3-A2-10-233 (SEQ ID NO 28), FOXp3-A2-10-152 (SEQ ID NO 29), FOXp3-A2-10-77 (SEQ ID NO 30), FOXp3-A2 -10-246 (SEQ ID NO 37), FOXp3-A2-9-68 (SEQ ID NO 18), FOXp3-A2-9-304 (SEQ ID NO 19), Foxp3-A24-10-87 (SEQ ID NO) 67), and Foxp3-A24-10-60 (SEQ ID NO 74).

This may mean the following sequences FOXp3-A24-9-363 (SEQ ID NO 3), FOXp3-A24-9-366 (SEQ ID NO 7), FOXp3-A24-9-190 (SEQ ID NO 9), FOXp3-A24-9-207 (SEQ ID NO 4), FOXp3-A24-9-332 (SEQ ID NO 5), FOXp3-A24-9-337 (SEQ ID NO 8), FOXp3-A24-10-114 ( SEQ ID NO 12), FOXp3-A2-9-390 (SEQ ID NO 15), FOXp3-A2-9-69 (SEQ ID NO 16), FOXp3-A2-9-252 (SEQ ID NO 17), FOXp3- A2-10-359 (SEQ ID NO 22), FOXp3-A2-10-263 (SEQ ID NO 24), FOXp3-A2-10-94 (SEQ ID NO 27), FOXp3-A2-10-233 (SEQ ID NO 28), FOXp3-A2-10-152 (SEQ ID NO 29), FOXp3-A2-10-77 (SEQ ID NO 30), FOXp3-A2-10-246 (SEQ ID NO 37), FOXp3-A2- 9-68 (SEQ ID NO 18), FOXp3-A2-9-304 (SEQ ID NO 19), Foxp3-A24-10-87 (SEQ ID NO 67), and Foxp3-A24-10-60 (SEQ ID NO) 74) is homologous to peptides derived from other molecules known to sensitize the human immune system. To rule out this possibility, a homology analysis was performed using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) for the list of peptide sequences, showing no significant sequence Source.

These results show the following sequence FOXp3-A24-9-363 (SEQ ID NO 3), FOXp3-A24-9-366 (SEQ ID NO 7), FOXp3-A24-9-190 (SEQ ID NO 9), FOXp3-A24-9-207 (SEQ ID NO 4), FOXp3-A24-9-332 ( SEQ ID NO 5), FOXp3-A24-9-337 (SEQ ID NO 8), FOXp3-A24-10-114 (SEQ ID NO 12), FOXp3-A2-9-390 (SEQ ID NO 15), FOXp3- A2-9-69 (SEQ ID NO 16), FOXp3-A2-9-252 (SEQ ID NO 17), FOXp3-A2-10-359 (SEQ ID NO 22), FOXp3-A2-10-263 (SEQ ID NO) 24), FOXp3-A2-10-94 (SEQ ID NO 27), FOXp3-A2-10-233 (SEQ ID NO 28), FOXp3-A2-10-152 (SEQ ID NO 29), FOXp3-A2-10 -77 (SEQ ID NO 30), FOXp3-A2-10-246 (SEQ ID NO 37), FOXp3-A2-9-68 (SEQ ID NO 18), FOXp3-A2-9-304 (SEQ ID NO 19) , Foxp3-A24-10-87 (SEQ ID NO 67), and Foxp3-A24-10-60 (SEQ ID NO 74) are unique and, within our knowledge, induce unintentional immunity against any unrelated molecule The possibility of a reaction is small.

In conclusion, Foxp3 is an antigen that has been used to target T-reg cells, and vaccines using these epitopes may be useful for immunotherapy. it works.

discuss

From the data in Figure 6, injection of each hFoxp3-252 and mFoxp3-252 peptide vaccine can induce epitope-specific CTL in vivo. Representing both Foxp3 epitope peptides can induce CTL against target cells expressing Foxp3 and corresponding major histocompatibility complex molecules. In other words, it is suggested that these CTLs may recognize and regulate T lymphocytes (T-reg). To assess this hypothesis, BALB/c mice were used to examine the in vivo anti-tumor effects of these Foxp3 epitope peptides. It was clearly shown to have an anti-tumor effect in mice inoculated with each of hFoxp3-252 and mFoxp3-252 peptide. These results strongly suggest that tumor growth may be inhibited by inhibiting T-reg in the local tumor microenvironment without even using TAA epitope phenopeptide inoculation. The inventors of the present invention believe that CTL against tumor cells is induced when the tumor is present in the body, however, T-reg is also induced by some immunosuppressive factors derived from tumor cells and inhibits anti-tumor effector cell function. Since the use of Foxp3 antigen determinant peptide vaccination can eliminate immunosuppression by killing or inhibiting T-reg, it can display anti-tumor effects without vaccinating the TAA epitope or using a strong adjuvant to stimulate the entire immune system. .

In this way, vaccination with hFoxp3-252 peptide (KLSAMQAHL) (SEQ ID NO 17) induced CTL and superior anti-tumor effects of mFoxp3-252 peptide (KLGAMQAHL) (SEQ ID NO 88) (Figures 5 and 6) ). From these results, we believe that inoculation of hFoxp3-252 peptide phase Immunological tolerance is avoided more efficiently than inoculation with mFoxp3-252 peptide. In other words, since the amino acid sequence of hFoxp3-252 differs from mFoxp3-252 in the third position, we believe that the hFoxp3-252 peptide is an "non-autoantigen" in vivo and can effectively induce CTL against T-reg.

Conclusions show that Foxp3 can be used as a novel target for cancer immunotherapy. Furthermore, these results strongly support the inoculation with the Foxp3 epitope, which inhibits the function of T-reg and should be used for cancer immunotherapy of many types of cancer cells.

Figure 1 shows photographs of the results of the IFN-γ ELISPOT assay for CTLs induced by peptides derived from Foxp3. Figure 1A, well numbers #2 and 7 were stimulated with Foxp3-A24-9-363 (SEQ ID NO 3), #1 and #6 were stimulated with Foxp3-A24-9-366 (SEQ ID NO 7), #5 Foxp3-A24-9-190 (SEQ ID NO 9) stimulates, and #7 is stimulated by Foxp3-A24-10-87 (SEQ ID NO 67) and Foxp3-A24-10-60 (SEQ ID NO 74), Effective IFN-γ production was shown compared to the control group. In Figure 1B, well number #4 is stimulated by Foxp3-A24-9-207 (SEQ ID NO 4), #6 is stimulated by Foxp3-A24-9-332 (SEQ ID NO 5), and #6 via Foxp3-A24- 9-337 (SEQ ID NO 8) stimulation, and #1, CTL stimulated by Foxp3-A24-10-114 (SEQ ID NO 12), showed potent IFN-γ production compared to the control group.

Figure 2 shows photographs of the results of the IFN-γ ELISPOT assay for CTLs induced by peptides derived from Foxp3. In Figure 2A, well number #2 Stimulated by Foxp3-A2-9-390 (SEQ ID NO 15), #2 stimulated by Foxp3-A2-9-69 (SEQ ID NO 16), #6 via Foxp3-A2-9-252 (SEQ ID NO 17) Stimulation, #4 via Foxp3-A2-10-359 (SEQ ID NO 22) stimulation, #7 via Foxp3-A2-263 (SEQ ID NO 24) stimulation, and #2 and #5 via Foxp3-A2-10-94 (SEQ ID NO 27) Stimulated CTL showed potent IFN-γ production compared to the control group. In Figure 2B, all wells were stimulated with Foxp3-A2-10-233 (SEQ ID NO 28), well numbers #6 and #7 were stimulated with Foxp3-A2-10-152 (SEQ ID NO 29), and #5 via Foxp3 -A2-10-77 (SEQ ID NO 30) stimulation, and #1 via Foxp3-A2-10-246 (SEQ ID NO 37) and Foxp3-A2-10-94 (SEQ ID NO 27) stimulated CTL, phase Effective IFN-γ production was shown compared to the control group.

In Figure 2C, well numbers #1, #2, #4, #5, #7, #9, #11, and #12 are stimulated by Foxp3-A2-9-390 (SEQ ID NO 15), #5 and # 11 stimulated by Foxp3-A2-9-304 (SEQ ID NO 19), #7 stimulated by Foxp3-A2-9-68 (SEQ ID NO 7), #12 via Foxp3-A2-9-252 (SEQ ID NO 17) The stimulated CTL showed potent IFN-γ production compared to the control group.

Figure 3 shows the expanded population of cells in positive wells and an IFN-γ ELISA assay was performed. In the 3A, B and C images, CTL cell lines (solid diamonds) stimulated by Foxp3-A02-9-390 (SEQ ID NO: 15) showed potent IFN-compares compared to the control group (closed squares). γ production. In Figure 3D, CTL cell lines (solid diamonds) stimulated with Foxp3-A02-9-252 (SEQ ID NO: 17) showed efficacy compared to the control group (solid squares). Energy production of IFN-γ.

In Figure 3E, a CTL cell line (solid diamond) stimulated with Foxp3-A24-10-60 (SEQ ID NO: 74) showed potent IFN-γ production compared to the control group (closed square). In Figure 3F, CTL cell lines (solid diamonds) stimulated by Foxp3-A02-10-94 (SEQ ID NO: 27) showed potent IFN-γ production compared to the control group (closed squares). In Figure 3G, CTL cell lines (solid diamonds) stimulated with Foxp3-A24-10-87 (SEQ ID NO: 67) showed potent IFN-γ production compared to the control group (closed square).

Figure 4 shows the specific CTL activity against target cells with endogenous expression of Foxp3 and HLA-A* 02 or 24. CTL cell lines induced by Foxp3-A02-9-390 (SEQ ID NO: 15) and Foxp3-A02-9-252 (SEQ ID NO: 17) for Foxp3 and HLA-A02 in Figures 4A and B Both transfected 293T showed highly specific CTL activity. On the other hand, for the control group, no significant specific CTL activity was shown. In Figure 4C, a CTL cell line induced by Foxp3-A02-9-252 (SEQ ID NO: 17) showed high specific CTL activity for 293T transfected with both Foxp3 and HLA-A24. On the other hand, for the control group, no significant specific CTL activity was shown.

Figure 5 shows the in vivo immunogenicity analysis of Foxp3-252_h and Foxp3-252_m peptides. On days 0 and 7, BALB/c mice were injected subcutaneously with IFA-binding peptide or with only IFA. On day 14, vaccinated mouse spleen cells were harvested and used as responsive daughter cells. The corresponding pulsed 1x10 ⁴ RLmalel cells (closed squares), or cells without pulsed peptides (open squares) were used as stimulator cells for the IFN-γ ELISPOT assay. In each experiment, Foxp3-252_h(A) and Foxp3-252_m(B) were vaccinated in 5 mice (M1-M5), and IFA was injected in 3 mice (N1-N3) without injection of peptides. As a control group.

Figure 6 shows the in vivo anti-tumor effect of vaccination with Foxp3 epitope peptide. On day 0, BALB/c mice were injected with 1x10 ⁵ 4T1 breast cancer cell lines. On days 3 and 10, IFA was injected in combination with Foxp3-252_h (-open circle-), IFA in combination with Foxp3-252_m (-solid square-), and IFA without binding peptide (-solid triangle-). Unvaccinated mice (-x-) were also prepared in this experiment as normal tumor growth in the control group. Significant differences in tumor growth inhibition were observed in the group inoculated with the Foxp3 epitope. *: P <0.01; **: P < 0.005.

Figure 7 shows the affinity test of the Foxp3-9-252 substitute for HLA molecules. In Figure 7A, the CTLs induced by Foxp3-9-252-WT recognize the cells of the Foxp3-9-252-9V peptide on the HLA-A2 molecule. The IFN-γ ELISPOT assay was performed using a Foxp3-9-252-WT peptide-induced CTL cell line as a responsive cell and using a pulse of Foxp3-9-252-WT or Foxp3-9-252-9V peptide. T2 cells act as stimulator cells. T2 cells pulsed without peptides were prepared as a control group. In Figure 7B, Foxp3-9-252-9V and Foxp3-9-252-WT showed similar affinity for HLA-A2 molecules. An IFN-γ ELISA assay was performed using a Foxp3-9-252-WT peptide-induced CTL cell line as a responsive cell (1×10 ⁵ cells/well) and using Foxp3-9-252-WT (-filled circles - ), Foxp3-9-252-9V (-open circle-) or HIV-A02 (-solid triangle-) peptide pulsed T2 cells as stimulator cells (1 x 10 ⁴ cells/well). The peptide pulse of the stimulator cells was incubated at various concentrations of peptide and peptide at 37 degrees Celsius for 2 hours.

In Figure 7C, CTL can be induced by stimulation with a Foxp3-9-252 substitution of the target HLA-A2 molecule. CTLs for all of the target HLA-A2 substituted peptides were generated according to the methods described in the Materials and Methods project. Wells Nos. 3 and 7 were stimulated with Foxp3-9-252-3M, Well No. 7 was stimulated with Foxp3-9-252-3L, and well numbered 8 was stimulated with Foxp3-9-252-9V. In the control group, IFN-γ production was shown. In Figure 7D, stimulating cells coated with Foxp3-9-252-WT peptide were recognized as CTLs induced by Foxp3-9-252-9V. A CTL cell line induced with Foxp3-9-252-9V peptide was used as a response to daughter cells. In this assay, T2 cells incubated with Foxp3-9-252-9V (-filled round-) or T2 cells incubated with Foxp3-9-252-WT (-open round-) peptide were used. T2 cells that were not incubated with the peptide (-open circle-) were used as stimulator cells (1 x 10 ⁴ cells/well).

<110>Tumor Therapy Science Co., Ltd. <120>FOXP3 peptide vaccine <130>ONC-A0620P <150>US 60/878,615 <151>2007-01-03 <150>US 60/896,472 <151>2007-03 -22 <160>103 <170>PatentIn version 3.4 <210>1 <211>1869 <212>DNA <213> Race <220><221>CDS<222>(189)..(1484)<400>1 <210>2 <211>431 <212>PRT <213>People <400>2 <210>3 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>3 <210>4 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>4 <210>5 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>5 <210>6 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>6 <210>7 <211>9 <212>PRT <213>Artificial sequence <220><223>Peptide vaccine <400>7 <210>8 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>8 <210>9 <211>9 <212>PRT <213>Artificial sequence <220><223>Peptide vaccine <400>9 <210>10 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>10 <210>11 <211>10 <212>PRT <213>Artificial 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sequence<220><223>Peptide vaccine <400>23 <210>24 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>24 <210>25 <211>10 <212>PRT <213>Artificial sequence <220><223>Peptide vaccine <400>25 <210>26 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>26 <210>27 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>27 <210>28 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>28 <210>29 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>29 <210>30 <211>10 <212>PRT <213>Artificial sequence <220><223>Peptide vaccine <400>30 <210>31 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>31 <210>32 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>32 <210>33 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>33 <210>34 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>34 <210>35 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>35 <210>36 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>36 <210>37 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>37 <210>38 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>38 <210>39 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide<400>39 <210>40 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>40 <210>41 <211>9 <212>PRT <213>Artificial sequence <220><223>Peptide vaccine <400>41 <210>42 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>42 <210>43 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>43 <210>44 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>44 <210>45 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>45 <210>46 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>46 <210>47 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>47 <210>48 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>48 <210>49 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>49 <210>50 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>50 <210>51 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>51 <210>52 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>52 <210>53 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>53 <210>54 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>54 <210>55 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>55 <210>56 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>56 <210>57 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>57 <210>58 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>58 <210>59 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>59 <210>60 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>60 <210>61 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>61 <210>62 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>62 <210>63 <211>10 <212>PRT <213>Artificial sequence <220><223>Peptide vaccine <400>63 <210>64 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>64 <210>65 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>65 <210>66 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>66 <210>67 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>67 <210>68 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>68 <210>69 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>69 <210>70 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>70 <210>71 <211>10 <212>PRT <213>Artificial 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sequence<220><223>Peptide vaccine <400>83 <210>84 <211>10 <212>PRT <213>Artificial sequence <220><223>Peptide vaccine <400>84 <210>85 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>85 <210>86 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>86 <210>87 <211>10 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>87 <210>88 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>88 <210>89 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>89 <210>90 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>90 <210>91 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>91 <210>92 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>92 <210>93 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>93 <210>94 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>94 <210>95 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>95 <210>96 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>96 <210>97 <211>9 <212>PRT <213>Artificial sequence <220><223>Peptide vaccine <400>97 <210>98 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>98 <210>99 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>99 <210>100 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>100 <210>101 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>101 <210>102 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>102 <210>103 <211>9 <212>PRT <213>Artificial sequence<220><223>Peptide vaccine <400>103

Claims (22)

A peptide comprising the amino acid sequence of SEQ ID NO: 17. A peptide having cytotoxic T cell inducing ability having less than 15 amino acids, wherein the peptide comprises an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 17, and (b) SEQ ID NO: 17, wherein one or two amino acids are substituted or added. The peptide according to claim 2, wherein the N-terminal second amino acid is phenylalanine, tyrosine, methionine, tryptophan or leucine. The peptide according to claim 2, wherein the C-terminal amino acid is phenylalanine, leucine, isoleucine, tryptophan, methionine or valine. The peptide of claim 2, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 95, 97 or 98. A pharmaceutical preparation for modulating a regulatory T cell (T-reg cell), comprising one of the peptides of any one of the above claims 1 to 5, or a polynucleotide encoding the peptide. The pharmaceutical preparation according to claim 6, which is for inhibiting proliferation of T-reg cells. The pharmaceutical preparation according to claim 6, which is for administration to an individual whose HLA antigen is HLA-A24 or HLA-A02. The pharmaceutical preparation according to claim 6 of the patent application, which is used for Inhibits the function of T-reg cells. The pharmaceutical preparation according to claim 6, which is for treating cancer. A pharmaceutical preparation as described in claim 10, which is a vaccine. The pharmaceutical preparation according to claim 10 or 11, which further comprises another victory in addition to the peptide of any one of claims 1 to 5 or a polynucleotide encoding the peptide. A peptide, the other peptide having the ability to induce cytotoxic T cells against cancer cells, or a polynucleotide encoding the other peptide. An exosome having a complex on its surface, the complex comprising an HLA antigen and the peptide of any one of claims 1 to 5. The exosome is as described in claim 13 wherein the HLA antigen is HLA-A24 or HLA-A02. The exosome is as described in claim 14, wherein the HLA antigen is HLA-A2402 or HLA-A0201. An in vitro method for inducing an antigen to present a cell which exhibits a cytotoxic T cell inducing ability by causing an antigen to exhibit cell contact with a peptide of any one of claims 1 to 5 or encoding the inclusion as The gene of the polynucleotide of the peptide is transferred to an antigen presenting cell. An in vitro method for inducing cytotoxic T cells by contacting an antigen presenting cell and a CD8-positive cell or a peripheral blood mononuclear leukocyte with a peptide of any one of claims 1 to 5 or by encoding versus The nucleic acid of the TCR subunit multi-peptide that binds to the peptide of any one of claims 1 to 5 of the patent application is transduced. An isolated cytotoxic T cell induced by the peptide of any one of claims 1 to 5. A cytotoxic T cell as claimed in claim 18, which is induced by the method of claim 17 of the patent application. An antigen presenting cell comprising a complex formed between an HLA antigen and a peptide of any one of claims 1 to 5. The antigen-presenting cell of claim 20, which is induced by the method of claim 16 of the patent application. A peptide, an immunologically active fragment thereof, or a polynucleotide encoding the peptide, according to any one of claims 1 to 5, for use in the preparation of a vaccine for regulating T-reg cells in a body.